Profiling anti-influenza activities of the pathogen recognition receptor RIG-I: Novel targets for antiviral intervention strategies

RIG-I是结合甲型流感病毒（FLUAV）的5 'ppp-dsRNA柄的细胞病毒传感器。然后RIG-I转换构象，并可以通过夹在锅柄上并分解聚合酶复合物来发挥直接的抗病毒作用，但通常它也会触发导致I型干扰素（IFN）产生的抗病毒信号传导。我们已经产生了RIG-I的突变体，尽管缺乏抗病毒信号，但它们可以减少病毒RNA的合成。这些RIG-I突变体的抗病毒机制将通过定义最低限度需要的结构域和进一步的修饰进行探索和优化。我们已经创建了携带一种这样的RIG-I突变体（K271 A）的小鼠，并将它们的FLUAV抗性与wt和RIG-I KO小鼠进行比较。帕潘立酮是一种FDA批准的抗精神病药，Patel & Kukol（Virology 2017）预测其与PB 2结合，显示其模拟RIG-I的直接抗病毒活性，因为它减少了PB 2与核衣壳的结合并减少了PR 8/34菌株的RNA合成。我们将在细胞系和原代人气道细胞中测试帕利哌酮对各种FLUAV毒株的作用，研究RIG-I在其抗病毒作用中的潜在参与，并评估其与RIG-I激动剂的相互作用。此外，我们确定了rRNA衍生的自我RNA片段作为新的RIG-I配体。这些RIG-I刺激物可以由OAS/ RNase L产生，OAS/ RNase L又可以被dsRNA和糖酵解中间体果糖-1，6-二磷酸（F16 bP）激活。糖酵解的内源性中间体和先天免疫之间的这种可能的联系将在OAS/RNase L敲除细胞中进行跟踪。此外，将研究巨噬细胞的代谢状态对RNase L活化、自身RNA片段产生和随后的RIG-I介导的抗病毒活性的影响。

RIG-I is a cellular virus sensor that binds the 5’ppp-dsRNA panhandle of Influenza A virus (FLUAV). RIG-I then switches conformation and can exert a direct antiviral effect by clamping onto the panhandle and disassembling the polymerase complex, but normally it also triggers antiviral signaling leading to type I interferon (IFN) production. We had generated mutants of RIG-I that reduce viral RNA synthesis despite being deficient in antiviral signaling. The antiviral mechanism of these RIG-I mutants will be explored and optimized by defining the minimally required domain and by further modifications. We already have created mice bearing one such RIG-I mutant (K271A), and will compare their FLUAV resistance with wt and RIG-I KO mice. Paliperidone, an FDA-approved antipsychotic predicted by Patel & Kukol (Virology 2017) to bind PB2, was shown to mimic the direct antiviral activity of RIG-I as it diminished the binding of PB2 to nucleocapsids and reduced RNA synthesis of the PR8/34 strain. We will test Paliperidone against a variety of FLUAV strains in cell lines and primary human airway cells, investigate a potential involvement of RIG-I in its antiviral effect, and evaluate its interplay with RIG-I agonists. Also, we identified rRNA-derived self-RNA fragments as new RIG-I ligands. These RIG-I stimulators can be generated by the OAS/ RNase L that in turn can be activated by dsRNA and the glycolysis intermediate Fructose-1,6-bisphosphate (F16bP). This possible link between an endogenous intermediate of glycolysis and innate immunity will be followed up in OAS/RNase L knockout cells. Moreover, the influence of the metabolic state of macrophages on RNase L activation, self-RNA fragment generation and subsequent RIG-I-mediated antiviral activity will be investigated.